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The transcriptional corepressor NAB2 inhibits NGF-induced differentiation of PC12 cells.

Qu Z, Wolfraim LA, Svaren J, Ehrengruber MU, Davidson N, Milbrandt J - J. Cell Biol. (1998)

Bottom Line: We found that stably transfected PC12 cells expressing high levels of NAB2 do not differentiate, but rather continue to proliferate in response to NGF.Inhibition of PC12 differentiation by NAB2 overexpression was confirmed using two additional experimental approaches, transient transfection, and adenoviral infection.Cotransfection with p21(WAF1) restored the ability of NAB2-overexpressing PC12 cells to differentiate in response to NGF.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

ABSTRACT
The PC12 pheochromocytoma cell line responds to NGF by undergoing growth arrest and proceeding to differentiate toward a neuronal phenotype. Among the early genetic events triggered by NGF in PC12 cells are the rapid activation of the zinc finger transcription factor Egr1/NGFI-A, and a slightly delayed induction of NAB2, a corepressor that inhibits Egr1 transcriptional activity. We found that stably transfected PC12 cells expressing high levels of NAB2 do not differentiate, but rather continue to proliferate in response to NGF. Inhibition of PC12 differentiation by NAB2 overexpression was confirmed using two additional experimental approaches, transient transfection, and adenoviral infection. Early events in the NGF signaling cascade, such as activation of MAP kinase and induction of immediate-early genes, were unaltered in the NAB2-overexpressing PC12 cell lines. However, induction of delayed NGF response genes such as TGF-beta1 and MMP-3 was inhibited. Furthermore, NAB2 overexpression led to downregulation of p21(WAF1), a molecule previously shown to play a pivotal role in the ability of PC12 cells to undergo growth arrest and commit to differentiation in response to NGF. Cotransfection with p21(WAF1) restored the ability of NAB2-overexpressing PC12 cells to differentiate in response to NGF.

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NAB2 inhibition of differentiation is prevented by the  Egr1 R1 domain. (A) PC12 cells were cotransfected with a lacZ  reporter plasmid at a 1:20 ratio with either control vector or  pCMVNAB2. 2 d after transfection, cells were exposed to NGF for  3 d, and stained for lacZ gene expression. Photographs are representative fields of lacZ-positive cells transfected with either non-recombinant (CONT) vector or pCMVNAB2 as indicated. (B)  PC12 cells were cotransfected with a lacZ reporter plasmid and  either NAB2, NAB1, NAB2 + Egr1 R1 domain, NAB2 + mutant  Egr1 R1 (I293F) or non-recombinant (CONT) vector as indicated.  The histogram shows the percentage of lacZ-positive cells that  remain undifferentiated after NGF treatment for 3 d beginning 2 d  after transfection. For each condition, >100 lacZ-positive cells  were counted. Cells with neurites longer than two cell body diameters were scored as morphologically differentiated. The data is  derived from four independent transfections. Error bars indicate  the standard deviation. (C) PC12 cells were infected with recombinant adenovirus AdGFP (CONT, top) or AdGFP NAB2 (bottom).  NGF was added 2 d after infection and cells were maintained for  an additional 5 d. The cells were visualized by phase-contrast (left)  or fluorescence (right) microscopy. The green fluorescent (GFP-positive) cells are those that have been infected with adenovirus.
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Figure 4: NAB2 inhibition of differentiation is prevented by the Egr1 R1 domain. (A) PC12 cells were cotransfected with a lacZ reporter plasmid at a 1:20 ratio with either control vector or pCMVNAB2. 2 d after transfection, cells were exposed to NGF for 3 d, and stained for lacZ gene expression. Photographs are representative fields of lacZ-positive cells transfected with either non-recombinant (CONT) vector or pCMVNAB2 as indicated. (B) PC12 cells were cotransfected with a lacZ reporter plasmid and either NAB2, NAB1, NAB2 + Egr1 R1 domain, NAB2 + mutant Egr1 R1 (I293F) or non-recombinant (CONT) vector as indicated. The histogram shows the percentage of lacZ-positive cells that remain undifferentiated after NGF treatment for 3 d beginning 2 d after transfection. For each condition, >100 lacZ-positive cells were counted. Cells with neurites longer than two cell body diameters were scored as morphologically differentiated. The data is derived from four independent transfections. Error bars indicate the standard deviation. (C) PC12 cells were infected with recombinant adenovirus AdGFP (CONT, top) or AdGFP NAB2 (bottom). NGF was added 2 d after infection and cells were maintained for an additional 5 d. The cells were visualized by phase-contrast (left) or fluorescence (right) microscopy. The green fluorescent (GFP-positive) cells are those that have been infected with adenovirus.

Mentions: To confirm that the results of NAB2 overexpression in stable cell lines was not due to the clonal selection process, we examined the ability of NAB2 to block NGF-induced PC12 differentiation in transient transfection experiments. PC12 cells were cotransfected with NAB2 and a CMV-driven lacZ reporter gene (to identify transfected cells). 2 d after transfection, cells were treated with NGF for 3 d. Approximately 70% of PC12 cells cotransfected with lacZ and NAB2 failed to develop neurites after 3 d of NGF treatment. In contrast, only 11% of cells cotransfected with lacZ and the non-recombinant control vector failed to develop neurites (Fig. 4, A and B).


The transcriptional corepressor NAB2 inhibits NGF-induced differentiation of PC12 cells.

Qu Z, Wolfraim LA, Svaren J, Ehrengruber MU, Davidson N, Milbrandt J - J. Cell Biol. (1998)

NAB2 inhibition of differentiation is prevented by the  Egr1 R1 domain. (A) PC12 cells were cotransfected with a lacZ  reporter plasmid at a 1:20 ratio with either control vector or  pCMVNAB2. 2 d after transfection, cells were exposed to NGF for  3 d, and stained for lacZ gene expression. Photographs are representative fields of lacZ-positive cells transfected with either non-recombinant (CONT) vector or pCMVNAB2 as indicated. (B)  PC12 cells were cotransfected with a lacZ reporter plasmid and  either NAB2, NAB1, NAB2 + Egr1 R1 domain, NAB2 + mutant  Egr1 R1 (I293F) or non-recombinant (CONT) vector as indicated.  The histogram shows the percentage of lacZ-positive cells that  remain undifferentiated after NGF treatment for 3 d beginning 2 d  after transfection. For each condition, >100 lacZ-positive cells  were counted. Cells with neurites longer than two cell body diameters were scored as morphologically differentiated. The data is  derived from four independent transfections. Error bars indicate  the standard deviation. (C) PC12 cells were infected with recombinant adenovirus AdGFP (CONT, top) or AdGFP NAB2 (bottom).  NGF was added 2 d after infection and cells were maintained for  an additional 5 d. The cells were visualized by phase-contrast (left)  or fluorescence (right) microscopy. The green fluorescent (GFP-positive) cells are those that have been infected with adenovirus.
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Related In: Results  -  Collection

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Figure 4: NAB2 inhibition of differentiation is prevented by the Egr1 R1 domain. (A) PC12 cells were cotransfected with a lacZ reporter plasmid at a 1:20 ratio with either control vector or pCMVNAB2. 2 d after transfection, cells were exposed to NGF for 3 d, and stained for lacZ gene expression. Photographs are representative fields of lacZ-positive cells transfected with either non-recombinant (CONT) vector or pCMVNAB2 as indicated. (B) PC12 cells were cotransfected with a lacZ reporter plasmid and either NAB2, NAB1, NAB2 + Egr1 R1 domain, NAB2 + mutant Egr1 R1 (I293F) or non-recombinant (CONT) vector as indicated. The histogram shows the percentage of lacZ-positive cells that remain undifferentiated after NGF treatment for 3 d beginning 2 d after transfection. For each condition, >100 lacZ-positive cells were counted. Cells with neurites longer than two cell body diameters were scored as morphologically differentiated. The data is derived from four independent transfections. Error bars indicate the standard deviation. (C) PC12 cells were infected with recombinant adenovirus AdGFP (CONT, top) or AdGFP NAB2 (bottom). NGF was added 2 d after infection and cells were maintained for an additional 5 d. The cells were visualized by phase-contrast (left) or fluorescence (right) microscopy. The green fluorescent (GFP-positive) cells are those that have been infected with adenovirus.
Mentions: To confirm that the results of NAB2 overexpression in stable cell lines was not due to the clonal selection process, we examined the ability of NAB2 to block NGF-induced PC12 differentiation in transient transfection experiments. PC12 cells were cotransfected with NAB2 and a CMV-driven lacZ reporter gene (to identify transfected cells). 2 d after transfection, cells were treated with NGF for 3 d. Approximately 70% of PC12 cells cotransfected with lacZ and NAB2 failed to develop neurites after 3 d of NGF treatment. In contrast, only 11% of cells cotransfected with lacZ and the non-recombinant control vector failed to develop neurites (Fig. 4, A and B).

Bottom Line: We found that stably transfected PC12 cells expressing high levels of NAB2 do not differentiate, but rather continue to proliferate in response to NGF.Inhibition of PC12 differentiation by NAB2 overexpression was confirmed using two additional experimental approaches, transient transfection, and adenoviral infection.Cotransfection with p21(WAF1) restored the ability of NAB2-overexpressing PC12 cells to differentiate in response to NGF.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

ABSTRACT
The PC12 pheochromocytoma cell line responds to NGF by undergoing growth arrest and proceeding to differentiate toward a neuronal phenotype. Among the early genetic events triggered by NGF in PC12 cells are the rapid activation of the zinc finger transcription factor Egr1/NGFI-A, and a slightly delayed induction of NAB2, a corepressor that inhibits Egr1 transcriptional activity. We found that stably transfected PC12 cells expressing high levels of NAB2 do not differentiate, but rather continue to proliferate in response to NGF. Inhibition of PC12 differentiation by NAB2 overexpression was confirmed using two additional experimental approaches, transient transfection, and adenoviral infection. Early events in the NGF signaling cascade, such as activation of MAP kinase and induction of immediate-early genes, were unaltered in the NAB2-overexpressing PC12 cell lines. However, induction of delayed NGF response genes such as TGF-beta1 and MMP-3 was inhibited. Furthermore, NAB2 overexpression led to downregulation of p21(WAF1), a molecule previously shown to play a pivotal role in the ability of PC12 cells to undergo growth arrest and commit to differentiation in response to NGF. Cotransfection with p21(WAF1) restored the ability of NAB2-overexpressing PC12 cells to differentiate in response to NGF.

Show MeSH
Related in: MedlinePlus